Analysis of Simian Immunodeficiency Virus-specific CD8<sup>+</sup> T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Lucas Gonzalez-Nieto; Aline Domingues; Michael Ricciardi; Martin J. Gutman; Helen S. Maxwell; Nuria Pedre&#241;o-Lopez; Varian Bailey; Diogo M. Magnani; Mauricio A. Martins

doi:10.3791/54881

サル免疫不全ウイルス特異的CD8の分析 +ペプチドMHC-I四量体染色によりアカゲザルにおけるT細胞

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Immunology and Infection

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JoVE Journal Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8⁺ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

サル免疫不全ウイルス特異的CD8の分析 +ペプチドMHC-I四量体染色によりアカゲザルにおけるT細胞

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December 23, 2016

DOI: 10.3791/54881-v

Lucas Gonzalez-Nieto¹, Aline Domingues¹, Michael Ricciardi¹, Martin J. Gutman¹, Helen S. Maxwell¹, Nuria Pedreño-Lopez¹, Varian Bailey¹, Diogo M. Magnani¹, Mauricio A. Martins¹

¹Department of Pathology,University of Miami

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Overview

This article presents an optimized protocol for enumerating and characterizing CD8 + T cells specific to the AIDS virus in rhesus macaques. The method allows for the quantification of antigen-specific CD8 T-cells in biological samples obtained ex vivo, providing insights into T-cell responses in various disease contexts.

Key Study Components

Area of Science

HIV immunology
T-cell immunology
Biomedical research

Background

CD8 + T cells play a crucial role in immune responses against viral infections.
Understanding the frequency of antigen-specific CD8 T-cells is vital for vaccine development.
Challenges exist due to incomplete knowledge of rhesus macaque MHC genetics.
Flow cytometric staining requires precise antibody application.

Purpose of Study

To enumerate and characterize CD8 positive T-cell populations against the Simian Immunodeficiency Virus.
To evaluate T-cell responses induced by vaccination or primary infection.
To provide a reliable method for studying CD8 T-cell responses in various disease settings.

Methods Used

Re-suspension of rhesus PBMC in R10 medium.
Flow cytometry for quantifying antigen-specific CD8 T-cells.
Direct analysis of biological samples without in vitro re-stimulation.
Characterization of T-cell populations generated by vaccination or infection.

Main Results

The method successfully quantifies antigen-specific CD8 T-cells in ex vivo samples.
It provides insights into the frequency of T-cell responses to vaccination and infection.
Facilitates the study of CD8 T-cell responses in multiple disease contexts.
Addresses challenges related to MHC genetics and antibody application.

Conclusions

This optimized protocol is a valuable tool for HIV immunology and broader biomedical research.
It enhances understanding of CD8 T-cell dynamics in response to viral infections.
The technique can be adapted for various research applications involving T-cell responses.

Frequently Asked Questions

What is the significance of CD8 + T cells in HIV research?

CD8 + T cells are crucial for controlling viral infections, including HIV, and understanding their responses can inform vaccine development.

How does this method differ from traditional techniques?

This method allows for the quantification of T-cells directly from biological samples without the need for in vitro re-stimulation, providing more accurate results.

What challenges do researchers face when using this protocol?

Researchers may struggle with the complexities of rhesus macaque MHC genetics and the need for precise antibody application during flow cytometry.

Can this technique be applied to other diseases?

Yes, the technique can be adapted to study CD8 T-cell responses in various disease settings beyond HIV.

What are the implications of this research?

The findings can enhance our understanding of immune responses and improve strategies for vaccine development against viral infections.

Is prior experience required to use this method?

While prior experience can be beneficial, detailed protocols and guidance can help new researchers successfully implement the technique.

ここでは、エイズウイルスに対するアカゲザルCD8 ⁺ T細胞を列挙し、特徴づけるための最適化されたプロトコルを提示します。この記事では、HIV免疫学の分野にだけでなく、CD8 ⁺ T細胞応答が疾患の転帰に影響を与えることが知られている生物医学研究の他の分野のみならず便利です。

このアッセイの全体的な目標は、ワクチン接種または一次感染によって生成されたサル免疫不全ウイルス特異的CD8陽性T細胞集団を列挙し、特徴づけることです。この方法は、ワクチン接種または感染によって誘導される抗原特異的CD8 T細胞の頻度など、T細胞免疫学分野における重要な質問に答えるのに役立ちます。この技術の主な利点は、in vitroでの再刺激を必要とせずに、ex vivoで直接得られた生体サンプル中の抗原特異的CD8 T細胞を定量できることです。

この手法の意味は、アカゲザルの切開特異的CD8 T細胞の評価や、複数の疾患設定におけるCDB T細胞応答の研究にまで及びます。一般に、この方法に不慣れな人は、アカゲザルのMHC遺伝学に関する知識が不完全であること、またフローサイトメトリー染色には正確な抗体の適用が必要であるため、苦労するでしょう。まず、アカゲザルPBMCをR10培地に再懸濁します。

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